Milk product enhanced in amino acids and peptides



Patented Aug. 25, 1953 MILK PRODUCT ENHANCED IN AIVHNO ACIDS AND PEPTIDES UNITED STATES PATENT OFFICE Delaware No Drawing. Application April 7, 1950, Serial No. 154,724

4 Claims.

1 This invention relates to a milk product enhanced in soluble, non-coagulable hydrolysis products of milk proteins (amino acids and peptides), to a lactose composition comprising such 2 Cystine can be synthesized from methionine in the human body, but methionine cannot. However, the excess of methionine in cows milk protein is insufficient to make good the deficiency protein hydrolysis products and adapted to be 5 of cystine in this way. Furthermore, there is used as an ingredient in such milk product, and some evidence to show that methionine in bovine to a method of preparing tlfie lactose ciompocficasein is ntoht ashiivailaltile for this purpose as tion. In one embodiment 0 our inven 1011 c previousy oug -Wi iams and Elvehjem, J. milk product and the lactose composition are Biol. Chem., 181,559 (1949).

selectively enhanced in tryptophzznledang sulfurm fTheflilollowing table gives the average content containing amino acids. Our me o o prepar- 0 me ionine and cystine as reported by all ining a lactose composition enhanced in the desired vestigators up to 1946 in terms of grams of the protein hydrolysis products, especially amino respective amino acids per 16 grams nitrogen acids and peptides, comprises proteolytically i. e. the respective amino acid content related to digesting whey, demineralizing the digested whey total protein content:

by contact with ion-exchange materials, thereby TABLE 1 simultaneously removing nitrogenous materials selectively from the whey, and recovering the G. amino acid. per 16 g nitrogen desired lactose product from the whey.

Our milk product is a highly nutritious food, 20 Cows- Human providing protein hydrolysis products in a readily g g assimilable form; as a liquid concentrate it can be heat-sterilized without substantial adverse Methionine" a 2 2 2 effect. Our lactose composition, as will be seen Cvsrine. .....l IZIIIIIIIIIIII j M from the following description, is particularly adapted to serve as an ingredient in such a milk 1 and Boning Arch Biochem" 10) 359 product and can be economically produced. Our 194 method of production permits demineralization t methionine and cysmne being sulfur-conwith ion-exchange resins without precipitation taining amino acids and methionine being On 01" clo of t e resin d in either UDfiOW vertible in the system to cystine, their combined l tl i h to improved replacements of avinagimyimay be compared in terms of mimn 8 Seam mo s 0 am no-acicl sulfur. (Met i mothers milk, attention has been directed to the 149.15 with 1 sulfur atom; cystin z ai /fi al. p b em f e 3 2233 513 2 gigg gg gn i f the with 25min; atotms.) The data in Table 1 then pro ems o 1 conver 0 ese erm Cows milk on the average contains about 2.8 S percent casein and 0.5 percent lactalbumin; the TABLE 2 corresponding figures for human milk are 0.5 per- Millimols amino-acid sulfur per 16 g. nitrogen cent casein and 1 percent lactalbumin. Since cows milk contains approximately twice as much Cows. Human total protein as human milk, it is ordinarily di- 40 Milk Milk luted to about 5 its original protein content for Prom infant feeding. When so diluted, however, the Methionine compositi n or it protein content is quite difier- Cy nus,, ;:::::::::::t:;::::::::::11:31:31; %:1 it ent from that of human milk. Measured in Tom terms of the amino-acid content of the respec- 3&1 tive proteins, the chief differences are a deficiency In h m t in cows milk of tryptophane and cystine, and a 25 3 i i is thus an excess of some somewhat greater content of methionine. All milk zg i g f as compared with these are essential amino acids. Accordingly, 5n nation 0 e same protein concenwhen cows milk is diluted 5 to approximately the protein content of human milk, the proteins in the diluted milk are deficient in tryptophane and cystine, as compared with human milk, but contain somewhat more methionine.

It is one object of our invention to provide a milk product enhanced in nutritionalhydrolysis products of milk proteins, especially amino acids and peptides.

It is a further object of our invention to provide a lactose composition containing such protein hydrolysis products and having a low-ash content, which is adapted to serve as a constituent of a reconstituted milk product.

It is an additional object of our inventicnto provide a milk product selectively enhanced in tryptophane and sulfur-containing amino-acids, and to provide a lactose composition suitable for use in preparing such a milk product.

It is a still further object of ourinvention to provide a simple and efllcient process for making such products.

Other objects and advantages will be apparent to those skilled in the art from the following description.

Lactose is usually prepared from Whey by a process involving deproteinizing' the whey by heat and acid coagulation, removal of the coagulum and crystallization of lactose from the deproteinized liquor.

If the whey is submitted to a proteolytic enzymic digestion, the soluble proteins may be converted completely to a non-coagulable form consisting of amino acids and peptides. We have discovered that if the thus treated whey is then subjected to a, demineralizing action by passing it successively over cationand anionexchange resins under controlled conditions, inorganic ions may be removed to an extent suf ficient to provide an acceptable low-ash prodnot, while substantial amounts of amino acids and peptides remain in solution, an eifluent being produced containing dissolved lactose together with amino acids and peptides. The demineralization may be so carried out as to produce a lactose product selectively enhanced in tryptophane and amino-acid sulfur, the ash content being reduced to a value acceptable in a reconstituted milk product. Such an efiiuent may be dried directly, as by spraying-drying, vacuum drum-dryin or the like, to produce a solid lactose product of low-ash content and containing the desired amino acids and peptides, or alternatively the efiluent may be evaporated to yield a liquid concentrate. Thus not only is enhancement in nutritional amino acids and peptides secured, but simultaneously lactose losses inherent in a crystallization process are eliminated. Furthermore, nutritional nitrogen is largely present in the readily assimilable form of uncondensed amino acids and peptides. As pointed out above the amino-acid content may be selectively enhanced in tryptophane and sulfur-bearing amino acids.

According to our invention we clarify and defat whey to remove any residual casein ilocs and fat globules that it may contain, and then submit it to a proteolytic fermentation. We prefer to use Has-activated papain as the ferment. but other proteolytic enzymes may be used such as pancreatic protease, protease from moldspepsin, and commercial proteolytic enzymes such asRHozyme" (Rohm and Haas, Philadelphia, Pa). If papain is used, it may be activated by other agents than HQS, such as HCNI ascorbic acid, etc., but B28 is our preferred activating agent.

Whenusing papain. we adjust the acidity of the why' within the range pH 5.0-7.2, preferably to about pH 5.5, and carry out the fermentation at an elevated temperature not above 70' C. till complete; e. g. with papain activated by H28 for about 30 minutes at about 37 C., about 2.5 to 6 hours at 60-65 C. are required. The fermented liquor is then heated to a higher temperature, preferably about 85 C., to inactivate the enzyme, cooled and filtered.

We then pass the filtrate successively over a cation-exchange resin and an anion-exchange resin. Various brands of such resin are avail able commerciallyand we do not limit our invention to particular brands, but We have had good results using "Amberlite IR-l05 (Rtihm and Haas, Philadelphia, Pa.) or "Nalcite MX (Dow Chemical Co., Midland, Mich.) as the cation exchanger and Duolite A-3 (Chemical Process Co., San Francisco, Calif.) as the anion exchanger.

We prefer to carry out the demineralization in steps. This may be done either in a mainand-scavenger system or in a countercurrent system.

In the former case the filtrate, containing lactose, amino acids, peptides and inorganic salts. is first passed through a main unit, comprising beds of cationand anion-exchange material, where the major part of the demineralization is effected and then through a similar smaller scavenger unit where the solution is finally demineralized to the desired extent. In each unit the relative sizes of the cationand anion-exchange beds are preferably chosen so that both beds become exhausted at nearly the same time when run in series, i. e. when a given volume of liquid is passed in turn through th cationand anion-exchange bed. The relative sizes of the main-and-scavenger units are also preferably chosen so that both units are exhausted to the desired level by the same volume of liquid passed successively through the main-and-scavenger units.

In practical operation conditions are so chosen as to effect demineralization to the desired extent, i. e. to not over 1% ash and preferably lower, while permitting passage of substantial amounts of the desired amino acids and peptides, especially tryptophane and the sulfurbearing amino acids, cystine and methionine. Ihis is achieved if the pH values of the eflluents are controlled according to the following table:

TABLE 3 Main-and-scavenger method p r P pmumt rn nnc rcmnge fen-ed eli iglgggt erred a A i an ,.fi i an Main cation .l. 5-i. 3 4. (l l. 7*2. 0 1.9 Mninanion 3.54.3 3.8 5.56.5 5.9 Scavenger cation 4. 14,3 4. 2 iii-4. 2 4.0 Scavenger anion 4. 2-5. 4 4. 7 5. 5-7. 0 1.0

scribed in the following examples, but these are to be taken as illustrative only and not as limiting our invention, the scope of which is defined in the appended claims.

EXAMPLE 1 One hundred and seventy-four liters cheese whey, pH 5.5, was digested for 1 hours at 66 C. with 54 g. Hrs-activated papain; more papain was added and the digestion continued for an additional 2 hours. The digest was then heated to 93 C. and filtered hot with the addition of a filter aid (Celite #505," Johns-Manville Corp, N. Y.). The filtrate was clear and contained no material that was heat-coagulable or precipitated by trichloroacetic acid.

A portion of the digest was then passed downfiow through main-and-scavenger deionizing units, in each unit being passed first through the cation bed and then through the anion bed.

Nalcite MX was used as the cation-exchanger and Duolite A-3" as the anion exchanger.

Condensed data for the deionizing steps follow:

TABLE 4 Main unit:

Cation bed volume ml 8800 Influent H 5.58 Eiliuent collected l 63 Eilluent pH, last portion 4.17 Efiluent pH. total eilluent 1.94

Anion bed volume ml 3500 Influent pH 1.94 Effluent collected l 56 Efiluent pH, last portion 3.82 Efiluent pH, total 5.95 Efiluent ash, solids basis- -percent 1.73

Scavenger unit:

Cation bed volume ml 1500 Influent pH 5.95 Eilluent collected l 40 Efiluent pH, last portion 4.27 Eilluent pH, total 4.18

Anion bed volume ml 750 Influent pH 4.18 Effluent collected l 39 Effluent pH, last portion 4.40 Efiluent pH, total 5.57

The solution remainded clear throughout the downflow demineralization and there was no precipitation on the resins. The volumes listed do not include sweetening-off water.

The finished solution was concentrated under vacuum to 25-30% solids content, filtered warm without filter aid and spray-dried. The yield was 1271 g. of a low-density nearly white, hygroscopic powder, having the following analysis:

TABLE 5 Moisture percent 1.53 Total nitrogen do 1.80 Protein equivalent (N x 6.25) do 11.2 Amino nitrogen do 0.35 Ash do 0.63 Tryptophane mg./g 1.38 Total Sulfur (method of Rosenheim &

Drummond) mg./g 0.67 Lactose monohydrate (by Munson-Walker) percent 89.64

The product was completely soluble in water to yield a clear solution.

EXAMPLE 2 Cheddar cheese whey was adjusted to pH 5.5 and digested with HiH-activated papain in an amount equal to 3% of the protein equivalent 0! the whey nitrogen; the digestion was carried out at about 66 C. until the digest gave no precipitate with trichloroacetic acid, requiring ordinarily 2 hours or somewhat longer. Alter inactivation at about -93 C., the digest was filtered with a filter aid to yield a sparkingiy clear greenish yellow solution.

This filtrate was doubie-demineralized by passing successively through two ion-exchange units, the first being partly-to-nearly-exhausted. and the second being fresh-to-partly-exhausted. When the first unit showed indications of breakthrough, it was taken off-stream for regeneration and a freshly regenerated unit shunted onstream. The latter then became the second unit and the former second unit became the first unit, in the direction of solution travel.

Each unit comprised a cation bed of 9175 ml. volume, cm. deep, containing Amberlite IR105" and an anion bed of 5300 ml. volume, cm. deep, containing "Duolite A-3. In each unit the solution passed through the cation bed and then through the anion bed. The cycle of operations for each unit comprised: rinse, backwash, regeneration, sweetening-on, demineralization to partial exhaustion in number 2 position, demineralizatlon to practical exhaustion (e. g. utilization of 85-98% capacity) in number i position, and sweetening-off. All eilluents containing 1% or more lactose were worked up; those containing less than 1 were discarded.

The demineralizing units were operated with digested whey through a number of cycles to bring the resins to equilibrium, after which eilluent was collected for working up. The ash content of the digested whey ranged from 0.50 to 0.65 mg./ml. (BA-10% on dry basis), the average ash throughout was 279 g. per cycle and the average pH of the efiluent was 6.9, the range bein from 6.6 to 7.4.

The collected eilluents were concentrated in a vacuum pan, roll-dried and pulverized. The product was a nearly white non-hygroscopic powder having the following analysis:

TABLE 6 Lactose, anhydrous (by Munson-Walker) per cent 90.5

The amino-acid-containing lactose product of our invention may advantageously be combined with other products to form a reconstituted milk product suitable for infant feeding, as a dietary adjunct for invalids and the ageing, and for similar uses. Such a product may advantageously contain skim cows milk, fat, vitamins, minerals and our lactose composition containing soluble non-coagulable hydrolysis products of milk proteins such as amino acids and peptides. The lactose content may also be supplied in part by the addition or ordinary crystalline edible lactose. Use of a mixture of lactose and our lactoseamino-acid peptide composition facilitates standardization and permits fixing the specification for total nitrogen at a figure lower than could be supplied by our composition.

A practicable range of composition for such a milk-base product is (by weight, dry basis) 25-35% skim milk solids, 25-35% emulsified edible fats, -25% edible lactose, and 20-45% of our lactose-amino-acid-peptide composition.

An example of such a product is the following:

TABLE '7 Approxi- Wet Bamate sis Dry llasis Percent Percent Skim cows milk (00. solids) 80. 6 27.3 Amino-acid-and-petpl.idccontaining lactose product 11.1 41. 8 Sodium marinate H 0,? 2. 6

Fat 7. 28. 2

iccithinhun .7

'lotnl 99.9 0.1.0

Balance-mincrals and vitamins.

The ingredients are blended and pasteurized in ways well known in the food industry. The resulting blend is concentrated and may be distributed either as a concentrated sterilized liquid of 23-28 Ct solid or as a powder containing preferably not over 2% moisture; with either form the addition of a suitable amount of water con verts the concentrate to a liquid milk-base food suitable for use as such.

EXAMPLE 4 The nutritive value of our food is indicated by rat-feeding experiments.

Each of three groups of 10 weanling rats of both sexes was fed one of the diet A, B and C ad libitum for 28 days; vitamin supplements were administered equally to all groups daily. Diet A was highly inadequate as to protein; diets B and C had a suboptimal and approximately equal protein content as estimated by nitrogen determination. Make-up of the diets is shown in the following table:

TABLE 8 Diet A Diet B Diet (7 Percent Percent Percent (rystullinv litl'lllSt. 57.0 .n. 51.84 Ainino-m-id-pc|itid\ -lurtosc product. 57. 0 CaseiiL. 8. 30 8. 30 i3. fill Lactaihumili. A 1.04 1.64 1.63 U. h. l. XII suit mix #2... 3.0 3.0 3.0 Ii(].0 10.0 30.0 Nitro murpfl. 1.32 2.14 2.08

The product of our invention used in diet B and designated above as Amino-acid-lactose The relative growth of the three groups of rats in terms of gain in weight is shown in the following table:

TABLE 10 Average Weight (lain per Rat of Groups Rccciving- Diet A Diet B Diet G. G. G. Initial wcighL... 52 52 62 Final weight" 51 Si 09 Gain l 29 17 Food consuined. .-i 77 99 80 Gain per g. food consumed 0. 20 0. l9 Gain per g. nitrogen consumed 13.7 9. 2

Diet A, high in lactose and low in protein, failed to produce growth.

Diet B containing our product thus proved acceptable (largest amount consumed per rat) and more nutritious and eificient than diet C (growth per rat, growth per g. food consumed, growth per g. nitrogen consumed). These results show that nitrogen in the form of amino acids and peptides in our product has better growth promotin properties than the same amount of casein nitrogen.

From the above description it will be seen that we have invented a novel and nutritious aminoacid-and-peptide-containing lactose product, especially adapted to be used as a constituent in reconstituted milk-base food products, and that we have provided a simple and economical process for manufacturing it. As will be clear from our disclosure to those skilled in the art, numerous modifications may be made without departing from the spirit of our invention and the scope of our claims.

We claim:

1. A process of producing an edible lactose product containing not over 1 percent by weight of ash on a solids basis and enhanced in soluble non-coagulable hydrolysis products of milk protein which comprises digesting whey in the presence of a proteolytic enzyme until substantially the entire protein content of the whey is converted to a form non-precipitable by trichloroacetic acid, and demineralizing the digested whey by contact successively with a cation-exchange resin and an anion-exchange resin, the amount of resins used and the volume of solution treated being so chosen as to reduce the ash content of the solution to not over 1 percent by weight on a solids basis and provide an eifluent having a pH between about 4.0 and about 7.0, whereby an eilluent is produced the solids content of which consists for the most part of lactose containing less than 1 percent by weight of ash and enhanced in soluble non-coagulable hydrolysis products of milk protein.

2. In combination with the process as defined in claim 1, the further step of concentrating the effluent at a temperature below its normal boiling point to a solids content of at least 20 percent by weight, to produce a concentrated edible lactose product containing less than 1 percent by weight of ash on a solids basis and enhanced in nutritional amino acids.

3. In combination with the process as defined in claim 1, the further steps of concentrating and drying the efiluent at non-deleterious temperatures to produce an edible solid lactose product containing not over 1 percent by weight of ash and enhanced in nutritional amino acids.

4. A process of producing an edible lactose product containing not over 1 percent by weight of ash on a solids basis and enhanced in nutritional amino acids and peptides which comprises:

9 digesting clarified and defatted whey, having a pH of about 5.5 in the presence of activated papain at a temperature in the range 50-70 C. until the proteins are substantially entirely converted to a form non-precipitable by trichloroacetic acid, 5

filtering the digest, demineralizing the filtrate by passing it in series through at least two demineralizing units, each unit comprising a bed of cation-exchange resin and a bed of anion-exchange resin, regulating the volume of solution treated with respect to the volume of the resin beds so that the eilluent from the last unit contains less than 1 percent by weight of ash on a solids basis and has a pH in the approximate range 4.0-7.0, and concentrating the eflluent by evaporation at a non-deleterious temperature below its normal boiling point.

FRANK H. TINKLER. REXFORD C. S'I'RIBLEY. JOHN G. KENNEDY.

10 References Cited in the file of this patent UNITED STATES PATENTS Number Name Date 1,041,896 stillwell Oct. 22, 1913- 1,085,380 Downham Jan. 2'7, 1914 2,465,906 Meade et al Mar. 29, 1949 2,477,558 Almy et a] Aug. 2, 1949 OTHER REFERENCES Associates of Lore A. Rogers, text "Fundamentals of Dairy Science, second edition 1935, pages 42, 48, 49, 64 to 68, 329.

Certificate of Correction Patent No. 2,650,166

August 25, 1953 FRANK H. TINKLER ET AL.

It is hereby certified that error appears in the rinted specification of the above numbered patent requiring correction as fol ows:

Column 3, line 45, for sprayingdrying read spray-drying; column 5, line 49, for remainded read remained; line 74, for H H-activated read H,;S-actioated; column 6, line 6, for sparkingly read sparklingly; line 38,

for throughout read throughput;

and that the said Letters Patent should be read as corrected above, so that the same may conform to the record of the case in the Patent Oflice.

Signed and sealed this 20th day of October, A. D. 1953.

ARTHUR W. CROCKER,

9 digesting clarified and defatted whey, having a pH of about 5.5 in the presence of activated papain at a temperature in the range 50-70 C. until the proteins are substantially entirely converted to a form non-precipitable by trichloroacetic acid, 5

filtering the digest, demineralizing the filtrate by passing it in series through at least two demineralizing units, each unit comprising a bed of cation-exchange resin and a bed of anion-exchange resin, regulating the volume of solution treated with respect to the volume of the resin beds so that the eilluent from the last unit contains less than 1 percent by weight of ash on a solids basis and has a pH in the approximate range 4.0-7.0, and concentrating the eflluent by evaporation at a non-deleterious temperature below its normal boiling point.

FRANK H. TINKLER. REXFORD C. S'I'RIBLEY. JOHN G. KENNEDY.

10 References Cited in the file of this patent UNITED STATES PATENTS Number Name Date 1,041,896 stillwell Oct. 22, 1913- 1,085,380 Downham Jan. 2'7, 1914 2,465,906 Meade et al Mar. 29, 1949 2,477,558 Almy et a] Aug. 2, 1949 OTHER REFERENCES Associates of Lore A. Rogers, text "Fundamentals of Dairy Science, second edition 1935, pages 42, 48, 49, 64 to 68, 329.

Certificate of Correction Patent No. 2,650,166

August 25, 1953 FRANK H. TINKLER ET AL.

It is hereby certified that error appears in the rinted specification of the above numbered patent requiring correction as fol ows:

Column 3, line 45, for sprayingdrying read spray-drying; column 5, line 49, for remainded read remained; line 74, for H H-activated read H,;S-actioated; column 6, line 6, for sparkingly read sparklingly; line 38,

for throughout read throughput;

and that the said Letters Patent should be read as corrected above, so that the same may conform to the record of the case in the Patent Oflice.

Signed and sealed this 20th day of October, A. D. 1953.

ARTHUR W. CROCKER,

Certificate of Correction Patent No. 2,650,166 August 25, 1953 FRANK H. TINKLER ET AL.

It is hereby certified that error appears in the {minted specification of the above numbered patent requiring correction as fol ows:

Column 3 line 45 for s raying-drying read eprayryz'ngcolumn 5 line 49, for remaindeii read remained line 74, for H,H-acti\;ated" read H,S-actz'vated; column 6, line 6, for sparkingly read sparklingly; line 38, for throughout read throughput; and that the said Letters Patent should be read as corrected above, so that the same may conform to the record of the case in the Patent Oflice.

Signed and sealed this 20th day of October, A. D. 1953.

ARTHUR W. CROCKER,

Assistant Commissioner of Patents. 

1. A PROCESS OF PRODUCTING AN EDIBLE LACTOSE PRODUCT CONTAINING NOT OVER 1 PERCENT BY WEIGHT OF ASH ON A SOLIDS BASIS AND ENHANCED IN SOLUBLE NON-COAGULABLE HYDROLYSIS PRODUCTS OF MILK PROTEIN WHICH COMPRISES DIGESTING WHEY IN THE PRESENCE OF A PROTEOLYTIC ENZYME UNTIL SUBSTANTIALLY THE ENTIRE PROTEIN CONTENT OF THE WHEY IS CONVERTED TO A FORM NON-PRECIPITABLE BY TRICHLOROACETIC ACID, AND DEMINERALIZING THE DIGESTED WHEY BY CONTACT SUCCESSIVELY WITH A CATION-EXCHANGE RESIN AND AN ANION-EXCHANGE RESIN, THE AMOUNT OF RESINS USED AND THE VOLUME OF SOLUTION TREATED BEING SO CHOSEN AS TO REDUCE THE ASH CONTENT OF THE SOLUTION TO NOT OVER 1 PERCENT BY WEIGHT ON A SOLIDS BASIS AND PROVIDE AN EFFLUENT HAVING A PH BETWEEN ABOUT 4.0 AND ABOUT 7.0, WHEREBY AN EFFLUENT IS PRODUCED THE SOLIDS CONTENT OF WHICH CONSISTS FOR THE MOST PART OF LACTOSE CONTAINING LESS THAN 1 PERCENT BY WEIGHT OF ASH AND ENHANCED IN SOLUBLE NON-COAGULABLE HYDROLYSIS PRODUCTS OF MILK PROTEIN. 